Studies on Wnt Genes in Neuroma-in-Continuity and During Nerve Regeneration
Arie C Van Vliet, MSc1; Martijn Tannemaat, MD, PhD2; Martijn JA Malessy, MD, PhD3; Fred De Winter, PhD1; Joost Verhaagen, PhD4; (1)Netherlands Institute for Neuroscience, Amsterdam, Netherlands, (2)University of Leiden, Leiden, Netherlands, (3)Department of Neurosurgery, Leiden University Medical Center, Leiden, Netherlands, (4)Neuroregeneration, Netherlands Institute for Neuroscience, Amsterdam, Netherlands
Introduction. A neuroma-in-continuity (NIC), formed following a human nerve lesion, impedes functional recovery. The mechanisms that underlie the formation of a NIC are poorly understood. Wnt ligand genes encode highly conserved glycoproteins with roles in development and tissue repair. Here we investigated Wnts and their receptors in NIC and in the injured rat sciatic nerve.
Materials and Methods. Wnt gene expression was investigated by microarray and qPCR. The biological activity of selected Wnt ligands was studied in embryonic and adult rodent dorsal root ganglion (DRG)-based repulsion and neurite outgrowth assays. Functional interference in a rat nerve injury and repair model was performed with lentiviral vectors encoding Wnt-inhibiting-factor 1 (WIF1) or in transgenic mice with Schwann cell specific mutation of Wnt5a.
Results. The expression of multiple genes of the Wnt family, including Wnt5a, are upregulated in NIC tissue. In the injured rat sciatic nerve, four Wnt-gene clusters with temporal expression profiles corresponding to specific phases of the regeneration process were identified. Schwann cells in the NIC and in the injured nerve are the source of Wnt5a, whereas the Wnt5a receptor Ryk is expressed by axons in the NIC. Wnt5a, Wnt5b and Wnt4 were chosen for functional studies because they are induced in the NIC formed following injury to a human nerve (Wnt5a) and in the injured rat nerve (Wnt5a, Wnt5b and Wnt4). Wnt5a, -5b and -4 promote neurite outgrowth of embryonic DRGs. Cultured naïve adult DRGs neurons did not significantly respond to Wnt5a, but displayed a clear trend towards enhanced neurite growth, whereas conditioned DRG neurons are inhibited by Wnt5a. This switch in responsiveness may be due to changes in Wnt-receptor composition. Indeed, DRG neurons without Ryk (obtained from Ryk0/0 mice), exhibit dramatically enhanced outgrowth in response to Wnt5a compared to wildtype DRGs, whereas overexpression of specific Wnt receptors (or combinations) either stimulates or impedes neurite growth in a DRG-like cellular screen. Collectively, these data suggest that the receptor composition affects the neurite outgrowth response of sensory neurites to Wnt ligands. Preliminary in vivo studies showed that functional perturbation of multiple ligands with the Wnt sequestering protein WIF1 or genetic deletion of a single Wnt gene (Wnt5a) in Schwann cells slows down axon regeneration.
Conclusion. Wnt signalling has a role in the regeneration process of an injured nerve. Increasing our understanding of these and other signalling pathways in the injured nerve is of importance to the development of nerve repair strategies.
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